optiPRM: A Targeted Immunopeptidomics LC-MS Workflow With Ultra-High Sensitivity for the Detection of Mutation-Derived Tumor Neoepitopes From Limited Input Material.

Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM.

T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells.

The advent of single-cell RNA sequencing (scRNAseq) has enabled in-depth gene expression analysis of several thousand cells isolated from tissues. We recently reported the application of scRNAseq toward the dissection of the tumor-infiltrating T-cell repertoire in human pancreatic cancer samples. In this study, we demonstrated that combined whole transcriptome and T-cell receptor (TCR) sequencing provides an effective way to identify tumor-reactive TCR clonotypes on the basis of gene expression signatures.

Low tumor interleukin-1β expression predicts a limited effect of adjuvant platinum-based chemotherapy for patients with completely resected lung adenocarcinoma: An identification and validation study.

Introduction And Objectives: Adjuvant platinum-based chemotherapy for completely resected non-small cell lung cancer is associated with modest improvement in survival; nevertheless, no validated biomarker exists for predicting the benefit or harm of adjuvant platinum-based chemotherapy.

Materials And Methods: We simultaneously measured 27 cytokines in operative tumor specimens from a discovery cohort (n = 97) by multiplex immunoassay; half of the patients received adjuvant platinum-based chemotherapy, and the other half were observed. We tested possible prognostic and predictive factors in multivariate Cox models for overall survival (OS) and relapse-free survival (RFS), and a tree-based method was applied to detect predictive factors with respect to RFS.

Prediction of tumor-reactive T cell receptors from scRNA-seq data for personalized T cell therapy.

The identification of patient-derived, tumor-reactive T cell receptors (TCRs) as a basis for personalized transgenic T cell therapies remains a time- and cost-intensive endeavor. Current approaches to identify tumor-reactive TCRs analyze tumor mutations to predict T cell activating (neo)antigens and use these to either enrich tumor infiltrating lymphocyte (TIL) cultures or validate individual TCRs for transgenic autologous therapies. Here we combined high-throughput TCR cloning and reactivity validation to train predicTCR, a machine learning classifier that identifies individual tumor-reactive TILs in an antigen-agnostic manner based on single-TIL RNA sequencing.

H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient.

H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells.

Transcriptome-based identification of tumor-reactive and bystander CD8 T cell receptor clonotypes in human pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is generally refractory to immune checkpoint blockade, although patients with genetically unstable tumors can show modest therapeutic benefit. We previously demonstrated the presence of tumor-reactive CD8 T cells in PDAC samples. Here, we charted the tumor-infiltrating T cell repertoire in PDAC by combining single-cell transcriptomics with functional testing of T cell receptors (TCRs) for reactivity against autologous tumor cells.

INTERCEPT H3: a multicenter phase I peptide vaccine trial for the treatment of H3-mutated diffuse midline gliomas.

Introduction: Diffuse midline gliomas (DMG) are universally lethal central nervous system tumors that carry almost unanimously the clonal driver mutation histone-3 K27M (H3K27M). The single amino acid substitution of lysine to methionine harbors a neoantigen that is presented in tumor tissue. The long peptide vaccine H3K27M-vac targeting this major histocompatibility complex class II (MHC class II)-restricted neoantigen induces mutation-specific immune responses that suppress the growth of H3K27M flank tumors in an MHC-humanized rodent model.

A H3K27M-targeted vaccine in adults with diffuse midline glioma.

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M diffuse midline glioma on a compassionate use basis.

T cell receptor dynamic and transcriptional determinants of T cell expansion in glioma-infiltrating T cells.

Background: Glioblastoma (GBM) is characterized by low numbers of glioma-infiltrating lymphocytes (GIL) with a dysfunctional phenotype. Whether this dysfunctional phenotype is fixed or can be reversed upon culturing is poorly understood. The aim of this study was to assess T cell receptor (TCR)-dynamics and -specificities as well as determinants of GIL expansion by sequencing-based technologies and functional assays to explore the use of GIL for cell therapy.

Salt-inducible kinase 3 protects tumor cells from cytotoxic T-cell attack by promoting TNF-induced NF-κB activation.

Background: Cancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies.

AMPLIFY-NEOVAC: a randomized, 3-arm multicenter phase I trial to assess safety, tolerability and immunogenicity of IDH1-vac combined with an immune checkpoint inhibitor targeting programmed death-ligand 1 in isocitrate dehydrogenase 1 mutant gliomas.

Introduction: Isocitrate dehydrogenase (IDH) mutations are disease-defining mutations in IDH-mutant astrocytomas and IDH-mutant and 1p/19q-codeleted oligodendrogliomas. In more than 80% of these tumors, point mutations in IDH type 1 (IDH1) lead to expression of the tumor-specific protein IDH1R132H. IDH1R132H harbors a major histocompatibility complex class II (MHCII)-restricted neoantigen that was safely and successfully targeted in a first-in human clinical phase 1 trial evaluating an IDH1R132H 20-mer peptide vaccine (IDH1-vac) in newly diagnosed astrocytomas concomitant to standard of care (SOC).

A vaccine targeting mutant IDH1 in newly diagnosed glioma.

Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H) tumours in syngeneic MHC-humanized mice.

Complete and long-lasting clinical responses in immune checkpoint inhibitor-resistant, metastasized melanoma treated with adoptive T cell transfer combined with DC vaccination.

Development of T cell-directed immune checkpoint inhibitors (ICI) has revolutionized metastatic melanoma (MM) therapy, but <50% of treated patients experience durable responses. This phase I trial (NCT01946373) investigates the safety/feasibility of tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) combined with dendritic cell (DC) vaccination in MM patients progressing on ICI. An initial cohort (5 patients) received TIL therapy alone to evaluate safety and allow for optimization of TIL expansion protocols.

Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells.

Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8 T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC).

The Outcome of TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic Growth Capacity between T-Cell Clones.

Purpose: During our efforts to develop tumor-infiltrating lymphocyte (TIL) therapy to counter the devastating recurrence rate in patients with primary resectable pancreatic ductal adenocarcinoma (PDA), we found that PDA TILs can readily be expanded and that the majority of resulting TIL cultures show reactivity against the autologous tumor. However, the fraction of tumor-reactive T cells is low. We investigated to which extent this was related to the expansion.

Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor-Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display.

Tumor-infiltrating lymphocytes (TIL) are considered enriched for T cells recognizing shared tumor antigens or mutation-derived neoepitopes. We performed exome sequencing and HLA-A02:01 epitope prediction from tumor cell lines from two HLA-A2-positive melanoma patients whose TIL displayed strong tumor reactivity. The potential neoepitopes were screened for recognition using autologous TIL by immunological assays and presentation on tumor major histocompatibility complex class I (MHC-I) molecules by Poisson detection mass spectrometry (MS).

Ipilimumab treatment decreases monocytic MDSCs and increases CD8 effector memory T cells in long-term survivors with advanced melanoma.

Ipilimumab has revolutionized malignant melanoma therapy, but a better understanding of the mechanisms behind treatment response and adverse effects is needed. In this work, the immune system of ipilimumab treated patients was monitored to investigate potential mechanisms of action that may correlate with treatment outcome. Blood samples from 43 advanced melanoma patients were taken before, during and at the end of treatment.

Identification of a tumor-reactive T-cell repertoire in the immune infiltrate of patients with resectable pancreatic ductal adenocarcinoma.

Purpose: The devastating prognosis of patients with resectable pancreatic ductal adenocarcinoma (PDA) presents an urgent need for the development of therapeutic strategies targeting disseminated tumor cells. Until now, T-cell therapy has been scarcely pursued in PDA, due to the prevailing view that it represents a poorly immunogenic tumor.

Experimental Design: We systematically analyzed T-cell infiltrates in tumor biopsies from 127 patients with resectable PDA by means of immunohistochemistry, flow cytometry, T-cell receptor (TCR) deep-sequencing and functional analysis of expanded T-cell cultures.

Next-generation TCR sequencing - a tool to understand T-cell infiltration in human cancers.

Tumour-infiltrating lymphocytes (TILs) are known to mediate potent anti-tumour activity. As T-cell-based therapies start to reach clinical practice, it becomes increasingly important to understand what characterizes a successful anti-tumour T-cell response and to exploit this knowledge for patient stratification. Next-generation sequencing of T-cell receptors (TCRs) promises to provide insights into the complexity of the tumour T-cell infiltrate that go beyond the phenotypic level.

Differential tumor infiltration by T-cells characterizes intrinsic molecular subtypes in breast cancer.

Background: Molecular subtypes of breast cancer and presence of tumor-infiltrating immune cells have both been implicated as important predictive and prognostic factors for improved risk stratification and treatment individualization of breast cancer patients. Their association, however, has not been studied in detail. The aim of this study was to evaluate the expression of the T cell markers CD8, FoxP3, CD3 and ζ-chain in molecular subtypes of the invasive margin and tumor center of breast cancer and corresponding sentinel nodes and to deduct prognostic information from these findings.

Melanoma and immunotherapy bridge 2015 : Naples, Italy. 1-5 December 2015.

MELANOMA BRIDGE 2015 KEYNOTE SPEAKER PRESENTATIONS Molecular and immuno-advances K1 Immunologic and metabolic consequences of PI3K/AKT/mTOR activation in melanoma Vashisht G. Y. Nanda, Weiyi Peng, Patrick Hwu, Michael A.

Non-classical HLA-class I expression in serous ovarian carcinoma: Correlation with the HLA-genotype, tumor infiltrating immune cells and prognosis.

In our previous studies, we have shown that patients with serous ovarian carcinoma in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A*02 genotype. This represent a stronger prognostic factor than loss or downregulation of the MHC class I heavy chain (HC) on tumor cells. In this study, we investigated the expression of the non-classical, immune tolerogenic HLA -G and -E on the tumor cells along with the infiltration of immune cells in the tumor microenvironment.